During the past 30 years a large number of antimicrobial agents of a number of different structural classes have been developed and used in treating bacterial infections. Important among these structural classes are the .beta.-lactams, glycopeptides, macrolides, quinolones, tetracyclines, and aminoglycosides. Of these, the .beta.-lactams include a large number of agents, and their mechanism of action has been studied in considerable depth.
.beta.-lactam antibacterial agents are generally effective antibacterial agents of relatively low toxicity (Donowitz and Mandell, 318 N. Engl. J. Med. 419-26 (1988)). .beta.-lactam agents contain the .beta.-lactam ring structure, and include e.g., penicillins, cephalosporins, carbacephems, carbapenems, penems, and monobactams. The .beta.-lactam agents kill bacteria by binding to specific target proteins in the cytoplasmic membranes of bacteria. When bound to the target proteins, the .beta.-lactam agents prevent biosynthesis of bacterial cell walls. These target proteins can be identified by their ability to covalently bind an isotopically-labeled .beta.-lactam, such as penicillin, and are termed penicillin-binding proteins (PBPs). The enzymatic functions of higher molecular weight PBPs are essential in the cross-linking of peptidoglycan in the bacterial cell wall. The antibacterial effects of the .beta.-lactam antibacterial agents arise from their ability to act as substrate analogs of the acyl-D-alanyl-D-alanine component of peptidoglycan. By acting as substrate analogs, the .beta.-lactam agents bind to and inhibit the enzymatic activity of high molecular weight PBPs, resulting in the inhibition of peptidoglycan synthesis. (Bryan and Godfrey, .beta.-lactam Antibodies: Mode of Action and Bacterial Resistance, Ch. 16 in Antibacterial Agents in Laboratory Medicine, 3rd Ed., Lorian ed. at 599-663 (1991)). The inhibition of synthesis of the rigid peptidoglycan layer usually causes the death of the bacteria because the rigid peptidoglycan layer is needed to maintain the integrity of the inner cytoplasmic membrane of the bacteria under conditions of low osmolarity. Thus, without a properly synthesized peptidoglycan layer, bacteria can swell and burst or, in cases where the PBPs are partially inhibited, the cells can exhibit filamentous growth leading to fragmentation.
Because of this ability to inhibit bacterial peptidoglycan synthesis, .beta.-lactam antibacterial agents have been highly effective and widely used to treat bacterial infections; however, mutant strains of bacteria which are resistant to the .beta.-lactam agents are encountered with increasing frequency. In particular, many strains of Staphylococcus aureus are developing increasing resistance to available antibacterial agents (the so-called methicillin resistant Staphylococcus aureus (MRSA)); such resistance is also present in strains of Staphylococcus epidermidis (MRSE).
There are three major mechanisms of bacterial resistance to .beta.-lactam agents. In many cases the resistance is due to the presence of a .beta.-lactamase which inactivates .beta.-lactam drugs by cleaving the .beta.-lactam ring. In addition, in gram-negative bacteria, resistance can be attained by a reduced cell permeability to .beta.-lactam agents. A third mode of .beta.-lactam resistance is alteration of the target of antibiotic action by structural modification or acquisition of a PBP which has a reduced binding affinity for the antibacterial agent. Although some of the organisms exhibiting this mode of resistance do not normally also produce a .beta.-lactamase, the MRSA often possess both resistance mechanisms.
Methicillin is a .beta.-lactamase stable .beta.-lactam antibacterial agent often used for the treatment of infections arising from .beta.-lactamase-producing strains of Staphylococcus aureus. However, the incidence of MRSA infections has become a serious problem (Chambers, Clin. Microb. Rev. 1:173-186 (1988); Neu, H., Science 257:1064-1073 (1992); De Lencastre et al., J. Antimicrob. Chemother. 33:7-24 (1994)). In fact, the current average incidence of MRSA in some large hospitals in the United States increased from 8% in 1986 to 40% in 1992.
Methicillin resistance is generally thought to be mediated by the resistance factor PBP2a, a 78-kDa PBP which has reduced affinity for .beta.-lactams, including methicillin. It is postulated that PBP2a compensates for the other inhibited PBPs in the presence of .beta.-lactam drugs, allowing continued peptidoglycan synthesis in the presence of .beta.-lactams (De Lencastre et al., supra, 1994).
In addition to the resistance factor PBP2a, there are several accessory factors involved in the expression of methicillin resistance in S. aureus. Included in this category are the fem gene products (Berger-Bachi and Kohler, FEMS Microbiol. Lett. 20: 305-309 (1983); Hartman and Tomasz, Antimicrob. Agents Chemother. 29(1): 85-92 (1986); Kornblum et al., Eur. J. Clin. Microbiol. 5: 714-718 (1986); Murakami et al., Antimicrob. Agents Chemother. 31: 1307-1311 (1987)), and the chr* gene product (Ryffel et al., Antimicrob. Agents Chemother. 38: 724-728 (1994); see also Lencaster & Tomosz, Antimicrob. Agents Chemother. 38:2590-2598 (1994)). Insertional inactivation of these genes in PBP2a-producing MRSA strains results in greater susceptibility to methicillin. The fem factors appear to be involved in the synthesis of peptidoglycan (Henze, et al., J. Bacteriol. 175(6): 1612-1620 (1993); De Jonge, et al., J. Bacteriol. 175(9): 2779-2782 (1993); Gustafson, et al., J. Bacteriol. 176(5): 1460-1467 (1994)).
Furthermore, MRSA have demonstrated the ability to rapidly develop resistance as new antibacterial agents become available. In the mid 1980s, new fluoroquinolone antimicrobial agents were developed, including ciprofloxacin, but the initial effectiveness of these compounds against staphylococci was short-lived. A study by the Centers for Disease Control showed that ciprofloxacin resistance of MRSA increased from less than 5% to greater than 80% within 1 year. (Neu, supra).
There are now MRSA strains which are susceptible to only a single class of clinically available antibacterial agents, the glycopeptides. Thus, there is a need for the development of new, efficient anti-MRSA treatments before resistance to glycopeptide antibacterial agents emerges in multi-resistant MRSA strains.
A mechanism for ampicillin resistance, similar to the mecA/PBP2a-mediated methicillin resistance in MRSA, has been observed in Enterococcus faecium. Such resistant strains produce a PBP (PBP5) which, like PBP2a in staphylococci, has low affinity for .beta.-lactams, and which is thought to carry on peptidoglycan synthesis when other PBPs have been inhibited by .beta.-lactams (Williamson et al., J. Gen. Microbiol. 131:1933-1940, 1985, Fontana et al., Antimicrob. Agents and Chemother. 38:1980-1983, 1994).
As with the .beta.-lactams, glycopeptides such as vancomycin and teicoplanin have been used for many years to treat a variety of Gram-positive bacterial infections. Until 1986 there were no reports of acquired resistance to these antibacterial agents. At that time, two research groups discovered high-level resistance in clinically important Enterococci (Leclercq et al., N. Engl. J. Med. 319:157-161 (1988); Uttley et al., Epidemiol. Infect. 103:173-181 (1989)). The resistance factors were carried on plasmids transmissible to other Gram-positive bacteria. Transmission of the resistance mechanism to Staphylococci is possible (and likely in the future) though no clinical isolates of S. aureus have yet been confirmed. However, resistance to glycopeptides in the Enterococci is now very common, and isolates of Enterococci exist which are virtually untreatable, either with glycopeptides or with other antibacterial agents.
The mechanism of antibacterial activity of glycopeptides in susceptible bacteria differs from the mechanisms of most other antibiotics; rather than being enzyme inhibitors, the glycopeptides bind to a critical precursor in the cell wall synthesis process, masking the precursor and blocking subsequent steps in the process. Specifically, the glycopeptide binds to two terminal alanine residues in a peptidoglycan pentapeptide.
The mechanism of glycopeptide resistance in Enterococci is then due to the production of an alternative precursor for the cell wall synthesis process. In the most common example, the two terminal alanines of the pentapeptide are replaced by an alanine and a lactate moiety. Existing glycopeptides do not bind effectively to that altered peptide, resulting in resistance to the antibiotics.
The genetic basis of resistance to glycopeptides has been determined (Arthur & Courvalin, Antimicrob. Agents Chemother. 37:1563-1571 (1993)). In the clinically most important enterococci, E. faecium and E. faecalis, two principal phenotypes are observed (VanA, VanB). Both mechanisms lead to vancomycin resistance, but teicoplanin may still be effective against VanB isolates. The clinically less important enterococci, E. gallinarum and E. casseliflavus exhibit a third resistance phenotype (VanC), which is constitutively expressed (rather than being inducible) and is presumed to be chromosomally carried (rather than being located on a transmissible extragenic element).
The above material is not admitted to be prior art to the pending claims but is provided only to aid the understanding of the reader.